Targeted expansion of qa-1-peptide-specific regulatory cd8 t cells to ameliorate arthritis

ABSTRACT

Nanoparticles to treat autoimmune diseases and HIV infection are provided. The nanoparticles comprise a biocompatible polymer and a complex, wherein the complex is a major histocompatibility complex (MHC) class I antigen E (HLA-E) linked to a peptide, and wherein the HLA-E-peptide complex is linked to the surface of the nanoparticle. The present invention also relates to methods for treating autoimmune diseases and HIV infection.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 61/707,357, filed Sep. 28, 2012, and 61/773,959, filed Mar. 7, 2013, the entire contents of which are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NIH Grants T32CA070083 and RO1 AI37562. Accordingly, the Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to nanoparticles and methods for treating autoimmune diseases.

BACKGROUND OF THE INVENTION

Achieving a balance between induction of protective immunity against pathogens and maintenance of self-tolerance is a central feature of the adaptive immune system. Although negative selection in the thymus removes the majority of clones that express T cell receptors (TCR) with high affinity for self-peptide MHC products, this process is incomplete. A significant fraction of mature peripheral T cells that respond to self-peptide-MHC complexes may differentiate into effector cells in the context of inflammatory stimuli (Bouneaud et al., 2000; Goldrath and Bevan, 1999; Slifka et al., 2003). Although this process is constrained by abortive or defective TCR signaling resulting in cellular elimination (AICD) or inactivation (Martin et al., 1999; Kearney et al., 1994), these cell-intrinsic mechanisms may not suffice to prevent the development of autoimmune disorders (Anderton et al., 2001; Panoutsakopoulou et al., 2001). There is increasing evidence that self-tolerance may also depend on inhibitory interactions between effector T cells and regulatory or suppressive cells (Littman and Rudensky, 2010). A regulatory subset of CD8+ T cells, termed CD8+ Treg cells, has been found to inhibit follicular T helper cell responses, which are essential for production of autoantibodies and formation of ectopic germinal centers (GC). However, the potential contribution of CD8+ Treg cells to the pathogenesis and treatment of autoimmune disease is not well understood. Due to the severity and breath of autoimmune diseases such as lupus and rheumatoid arthritis, there is a great need for effective treatments of such diseases.

SUMMARY OF THE INVENTION

The present invention, in one aspect, relates to the development of strategies based on in vivo and in vitro expansion and activation of the CD8+ Treg cells for the treatment of autoimmune diseases.

In some aspects provided, is a composition for a nanoparticle comprising a biocompatible polymer and a complex, wherein the complex is a major histocompatibility complex (MHC) class I antigen E (HLA-E) linked to a peptide, and wherein the HLA-E-peptide complex is linked to the surface of the nanoparticle. In certain embodiments, at least 4 units, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 units of the HLA-E-peptide complex are linked together to form a moiety and the moiety is linked to the surface of the nanoparticle. In certain embodiments of the invention, the HLA-E-peptide complex of this nanoparticle composition comprises biotin, and the complexes are linked together through biotin-avidin interaction to form the moiety. In certain embodiments of the invention, the peptide linked to the HLA-E is selected from the group consisting of Hsp60_(p216), B7sp and FL9. In certain embodiments, the HLA-E-peptide complex or the moiety is linked to the surface of the nanoparticle via PEGylation.

In certain embodiments, the peptide of this complex is linked to HLA-E via a flexible linker. In certain embodiments, wherein the peptide is linked to HLA-E via a flexible linker, this linker is a Gly-Ser linker.

In certain embodiments, the heavy chain and light chain (β-2 microglobulin) of the HLA-E are linked via a flexible linker. In certain embodiments, wherein the heavy chain and light chain (β-2 microglobulin) of the HLA-E are linked via a flexible linker, the flexible linker is a Gly-Ser linker.

In certain embodiments, the biocompatible polymer of the nanoparticle is selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), chitosan, and chitosan-PEG. In certain embodiments of the nanoparticle composition, a low releasing dose of IL-15 is incorporated into the nanoparticle.

In other aspects provided, is a method for treating an autoimmune disease comprising: administering to a subject in need of such treatment nanoparticles, as described in the embodiments above, in an amount effective to ameliorate a symptom of the autoimmune disease. In certain embodiments of the invention, the autoimmune disease treated by the administration of nanoparticles is selected from a group consisting of systemic lupus erythematosus, chronic graft versus host disease, rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis, inflammatory bowel disease, Sjogren's syndrome, Graves disease, Crohn's disease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome, monoclonal gammopathy of undetermined origin, POEMS syndrome, myeloma, macroglobulinemia, and cold agglutinin disease.

In other aspects provided, is a method for treating an autoimmune disease comprising: contacting dendritic cells isolated from a subject in need of such treatment with Hsp60_(p216) peptide to generate Hsp60_(p216)-loaded dendritic cells; and administering to the subject the Hsp60_(p216)-loaded dendritic cells in an amount effective to ameliorate a symptom of the autoimmune disease. In certain embodiments the dendritic cells isolated from a subject in need of such treatment are contacted with Hsp60_(p216) for 2, 4, 6, 8, 12, 16, 18 or 24 hours to generate Hsp60_(p216)-loaded dendritic cells. In certain embodiments of the invention, this method is used to treat autoimmune diseases selected from the group consisting of systemic lupus erythematosus, chronic graft versus host disease, rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis, inflammatory bowel disease, Sjogren's syndrome, Graves disease, Crohn's disease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome, monoclonal gammopathy of undetermined origin, POEMS syndrome, myeloma, macroglobulinemia, and cold agglutinin disease.

In other aspects provided, is method for treating an autoimmune disease comprising: isolating from a subject in need of such treatment CD8+ T cells that bind Hsp60_(p216) peptide; growing the isolated cells in a culture medium containing IL-15C until the number of CD8+ T cells that bind Hsp60_(p216) peptide increases to at least 3-5% of CD8+ T cells, thereby producing a population of cells enriched with CD8+ T cells that bind Hsp60_(p216) peptide; and administering CD8+ T cells that bind Hsp60_(p216) peptide from the described population of cells to the subject in an amount effective to ameliorate a symptom of the autoimmune disease. In certain embodiments, the step of isolating CD8+ T cells that bind Hsp60_(p216) peptide comprises sorting a sample containing T cells obtained from the subject into CD8+ T cells that bind Hsp60_(p216) peptide using a fluorescently labeled moiety having at least 4 units of a complex, wherein the complex is a major histocompatibility complex (MHC) class I antigen E (HLA-E) linked to Hsp60_(p216) peptide. In certain embodiments, the HLA-E-peptide complex described comprises biotin, and at least 4 such complexes associate through biotin-avidin interaction to form the moiety. In certain embodiments, avidin is fluorescently labeled. In certain embodiments, the fluorescent label used to label avidin is Phycoerythrin (PE). In certain embodiments, the population of cells enriched with CD8+ T cells that bind Hsp60_(p216) peptide is further enriched using anti-R-Phycoerythrin (PE) microbeads. In certain embodiments, the autoimmune disease treated through these steps is selected from the group consisting of systemic lupus erythematosus, chronic graft versus host disease, rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis, inflammatory bowel disease, Sjogren's syndrome, Graves disease, Crohn's disease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome, monoclonal gammopathy of undetermined origin, POEMS syndrome, myeloma, macroglobulinemia, and cold agglutinin disease.

In some aspects provided, is a method for treating HIV infection. The method comprises administering to a subject in need of such treatment nanoparticles described herein in an amount effective to ameliorate a symptom of the HIV infection.

In some aspects provided, is a method for treating HIV infection. The method comprises contacting dendritic cells isolated from a subject in need of such treatment with Hsp60_(p216) peptide to generate Hsp60_(p216)-loaded dendritic cells; and administering to the subject the Hsp60_(p216)-loaded dendritic cells in an amount effective to ameliorate a symptom of the HIV infection. In certain embodiments, the dendritic cells are contacted with Hsp60_(p216) for 2, 4, 6, 8, 12, 16, 18 or 24 hours to generate Hsp60_(p216)-loaded dendritic cells.

In some aspects provided, is a method for treating HIV infection comprising: isolating from a subject in need of such treatment CD8+ T cells that bind Hsp60_(p216) peptide; growing the isolated cells in a culture medium containing IL-15C until the number of CD8+ T cells that bind Hsp60_(p216) peptide increases to at least 3-5% of CD8+ T cells, thereby producing a population of cells enriched with CD8+ T cells that bind Hsp60_(p216) peptide; and administering CD8+ T cells that bind Hsp60_(p216) peptide from the population of cells to the subject in an amount effective to ameliorate a symptom of the HIV infection. In certain embodiments, the step of isolating CD8+ T cells that bind Hsp60_(p216) peptide comprises sorting a sample containing T cells obtained from the subject into CD8+ T cells that bind Hsp60_(p216) peptide using a fluorescently labeled moiety having at least 4 units of a complex, wherein the complex is a major histocompatibility complex (MHC) class I antigen E (HLA-E) linked to Hsp60_(p216) peptide. In certain embodiments, the HLA-E-peptide complex described comprises biotin, and at least 4 such complexes associate through biotin-avidin interaction to form the moiety. In certain embodiments, avidin is fluorescently labeled. In certain embodiments, the fluorescent label used to label avidin is Phycoerythrin (PE). In certain embodiments, the population of cells enriched with CD8+ T cells that bind Hsp60_(p216) peptide is further enriched using anti-R-Phycoerythrin (PE) microbeads.

Each of the embodiments and aspects of the invention can be practiced independently or combined. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”, “comprising”, or “having”, “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

These and other aspects of the inventions, as well as various advantages and utilities will be apparent with reference to the Detailed Description. Each aspect of the invention can encompass various embodiments as will be understood.

All documents identified in this application are incorporated in their entirety herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is graph showing that selective expansion of Qa-1-restricted CD8+ Treg inhibits arthritis. Arthritis was induced in C57BL/6 mice as described in Methods. Irradiated LPS-activated Kb^(−/−)Db^(−/−) bone marrow-derived DC pre-loaded with or without indicated peptides was injected subcutaneously at the tail base at d27, d36 and d49 ( ). Arthritis scores are shown; 5-6 mice per group. Group (DC alone) versus group (Hsp60p216-DC), *P<0.05.

FIG. 1B is a plots showing the selective expansion of Qa-1-restricted CD8 Treg, where CII-immune B6 mice were immunized with Kb^(−/−)Db^(−/−)DC loaded with or without the indicated peptide as described in FIG. 1A. Qdm- or Hsp60p216-CD8 cells from draining lymph nodes were analyzed at d42 by staining with RA.Qa-1-Qdm-tetramer or WT.Qa-1-Hsp60p216-tetramer, respectively. Representative FACS plots are shown.

FIG. 2A is a plot showing that selective expansion of Qa-1-restricted CD8+ Treg inhibited arthritis. Hsp60p216-tet+ and Hsp60p216-tet− CD8 cells were sorted from CII-immune B6 mice before incubation in IL-15C (10 ng ml-1)*10d. These cells were then incubated with Hsp60p216 tetramer and enriched by anti-PE microbeads. FACS analysis of tetramer staining of these cells indicated that incubation of CD8 cells that were tet+ but not tet− resulted in a substantial number of Hsp60p216 tet+ cells.

FIG. 2B is a graph showing the transfer of Qa-1-Hsp60_(p216) tetramer⁺ CD8 T cells. The Hsp60p216-tet+ or Hsp60p216-tet− fraction of CD8 cells was transferred into Rag2^(−/−)Prf1^(−/−) mice along with CD4 and B cells from arthritic mice. Arthritis was induced as described in Methods. Arthritis scores are shown; 3 mice per group.

FIG. 2C is a bar graph showing anti-mouse CII IgG titers at d30; 3 mice per group. Group (tet− CD8) versus group (tet+ CD8), *P<0.05.

FIG. 2D is a bar graph showing anti-mouse CII IgG titers at d30. Here Qdm-tet+ or Qdm-tet− CD8 cells were transferred into Rag2^(−/−)Prf1^(−/−) mice along with CD4 and B cells from arthritic mice.

FIG. 2E is a bar graph showing selective expansion of Qa-1-restricted CD8 Treg. Hsp60p216-tet+ CD8 cells were enumerated at d34 after adoptive transfer as described in FIG. 2B.

FIG. 3 shows the amino acid sequence of human IL-15 (SEQ ID NO: 3; NCBI Reference Sequence: NP_(—)000576.1).

DETAILED DESCRIPTION OF THE INVENTION

A subpopulation of CD8+ regulatory T cells is essential for maintenance of self-tolerance and prevention of autoimmune disease (WO 2012/054509 incorporated by reference herein). These CD8+ Treg cells are programmed to suppress rather than activate immunity and represent an essential regulatory element of the immune response and a guarantor of self-tolerance. The present invention, in one aspect, relates to the development of strategies based on in vivo and in vitro expansion and activation of the CD8+ Treg cells for the treatment of autoimmune diseases.

The specialized regulatory CD8+ T cells selectively suppress CD4+ follicular helper T cell (TFH) activity through perforin-dependent lysis by recognition of class Ib major histocompatibility complex (MHC) peptide Qa-1 (mouse homolog of human leukocyte antigen E (HLA-E)) expressed at the surface of TFH cells and dampen autoantibody responses. Qa-1, the mouse homolog of human leukocyte antigen E (HLA-E), forms a heterodimer with β₂-microglobulin that binds to and presents peptides derived from self or foreign proteins after deliberate immunization or infection (Lo et al., 1999; Lo et al., 2000; Sullivan et al., 2002).

Three peptides are presented by Qa-1 (HLA-E): Qdm (B7sp in humans), Hsp60_(p216) and FL9. Qa-1 heterodimers containing peptides derived from MHC class Ia leader sequences, called Qdm (for Qa-1 determinant modifier), bind to nonclonally distributed CD94-NKG2A receptors expressed by natural killer (NK) cells and a subpopulation of CD8+ T cells. The functional consequence of Qa-1/Qdm-NKG2A interactions is generally inhibition of NK or CD8 cytolytic activity (Moser et al., 2002). Hsp60_(p216) peptide (GMKFDRGYI; SEQ ID NO: 1) contains residues 216-224 of the 60 kD heat shock protein (Hsp). The Hsp60_(p216) peptide can efficiently replace Qdm peptides under conditions of cellular activation and stress. A third peptide that is presented by Qa-1 under conditions of defective antigen processing in the endoplasmic reticulum (ERAAP dysfunction) is the highly conserved peptide FL9, FYAEATPML (SEQ ID NO: 2), which is derived from the conservative Fam49b gene. Recognition of Qa-1/FL9 by CD8 T cells may serve to eliminate abnormal cells that have defects in antigen presentation.

One of the potential platforms for specific expansion of Qa-1-restricted or HLA-E-restricted CD8+ Treg is based on vaccination or treatment with peptide-Qa-1(HLA-E)-coated nanoparticles (p-Qa-1(HLA-E)-NP). Administration of nanoparticles conjugated with Qa-1 tetramers without co-stimulatory activity is competent to stimulate Qa-1-peptide-specific CD8 T cells, since CD8+ Treg are memory cells according to function and phenotype. Unlike naïve CD8 cells, memory cells can be stimulated to expand by antigen presented without co-stimulation.

Accordingly, aspects of the invention involve nanoparticles (NP) comprising a biocompatible polymer and a complex of MHC class I antigen E (HLA-E) linked to a peptide. The HLA-E-peptide complex is linked to the surface of the nanoparticle to ensure efficient exposure to TCR on CD8 cells. To enhance the efficiency of p-Qa-1(HLA-E)-NP-mediated binding and expansion of CD8 Treg in vivo, in some embodiments, at least 4 units, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 units of the HLA-E-peptide complex are linked together to form a moiety and the moiety is then linked to the surface of the nanoparticle. In some embodiments, the HLA-E-peptide complex comprises biotin, and the complexes are linked together through biotin-avidin interaction to form the moiety. The moiety is conjugated to the carboxyl groups on the NP surface using carbodiimide coupling chemistry (EDC/NHS) to generate p-Qa-1 (HLA-E)-NP particles.

In some embodiments, the HLA-E-peptide complex or the moiety is linked to the surface of the nanoparticle via PEGylation. PEGylation of NP helps prevent internalization of p-Qa-1-NP by phagocytes. Covalent attachment of p-Qa-1(HLA-E) to the distal end of individual PEG molecules rather than to the NP core enables efficient exposure to TCR on CD8+ cells. Alternatively, low molecular-weight chitosan (LMWC) can be adopted as an alternative surface coating method. LMWC also provides hydrophilic layers on NP surface similar to PEG, reducing opsonization and phagocytic uptake of PLGA-LMWC by macrophages (Amoozgar Z. Mol Pharm 2012, Mochizuki M. FASEB 2003).

The NP can be synthesized using any biocompatible polymer, including but not limited to, poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), chitosan, and chitosan-PEG. In some embodiments, PLGA is employed to synthesize the NP for the delivery of p-Qa-1(HLA-E). PLGA is an FDA approved drug delivery reagent and displays favorable features among other available NP particles (Fe304-PEG, Quantum dots, or Liposomes), including controllable biodegradability, excellent biocompatibility, and high safety. PLGA-b-PEG copolymers can be synthesized by direct conjugation of PLGA-COOH with NH2-PEG-COOH, as described by Cheng et al. (Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery. Biomaterials 28, 869-876 (2007)). The carboxyl group in the copolymer is located at the terminal end of the hydrophilic group allowing availability for surface chemistry.

In some embodiments, the peptide is linked to the HLA-E via a flexible linker. In some embodiments, the heavy chain and light chain (β-2 microglobulin) of the HLA-E are linked via a flexible linker. It is understood and herein contemplated that any linker known by those of skill in the art can be used to make the disclosed NP. The choice of which linker to use can be determined by those of skill in the art based on the desired length and flexibility of the linker. Thus, in some embodiments, the linker can be any short peptide sequence as long as it is hydrophilic and forms a flexible linker by not building a secondary structure that interferes with the main domain structures. In some embodiments, glycine-serine, the most commonly used flexible linker for the production of recombinant single chain protein composed of different moieties, is used. The Gly-Ser linker can be but is not limited to (Gly4Ser)n or (Gly3Ser)n. Thus, for example, the linker can be G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, (G4S)7, (G4S)8, (G4S)9, (G4S)10, G3S, (G3S)2, (G3S)3, (G3S)4, (G3S)5, (G3S)6, (G3S)7, (G3S)8, (G3S)9, (G3S)10. It is further understood that the linkers used can be a combination of chemical and gly-ser linkers. For example, the linker between the peptide and the HLA-E can be a chemical linker and the linker between the heavy and light chains of the HLA-E can be a Gly-Ser linker. It is further understood that the for greater flexibility, a longer linker is used; whereas, a shorter linker is used for application where more rigidity is needed. Typically, the linker between the peptide and the HLA-E is 10-20 amino acids long, while the linker between the heavy and light chains of the HLA-E is 15-25 amino acids long. Specifically contemplated herein are NP wherein the linker between the peptide and the β-2 microglobulin of the HLA-E is composed of 15 amino acids [(G45)3] and β2-microglobulin and the heavy chain of HLA-E is connected by a linker composed of 20 amino acids [(G45)4].

CD8+ Treg cells depend on IL-15 for activation of suppressive activity. Thus, in some embodiments, a low releasing dose of IL-15 is incorporated into the nanoparticle. The low releasing dose of IL-15 helps promote the expansion of Qa-1(HLA-E)/peptide specific CD8 Treg cells upon stimulation by p-Qa-1(HLA-E). The amino acid sequence of human IL-15 is represented by NCBI Reference Sequence: NP_(—)000576.1 (SEQ ID NO: 3). IL-15 polypeptides also include fragments of IL-15, such as amino acids 49-162 of SEQ ID NO:3, which has previously been characterized as a mature form of IL-15 derived by proteolytic cleavage of a leader sequence from the polypeptide of NP_(—)000576.1, and other fragments that retain the biological activity of IL-15 are encompassed by the term IL-15.

The nanoparticles described herein can be used to treat autoimmune diseases. Accordingly, aspects of the invention involve a method for treating an autoimmune disease by administering to a subject in need of such treatment nanoparticles described herein in an amount effective to ameliorate a symptom of the autoimmune disease. In some embodiments, the peptide in the nanoparticles is Hsp60_(p216) Other aspects of the invention involve a method for treating an autoimmune disease by administering to a subject in need of such treatment a complex comprising a peptide conjugated to any biocompatible polymer, such as, but not limited to poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), chitosan, and chitosan-PEG. In some embodiments, the peptide is Hsp60_(p216) In some embodiments, the complex comprises Hsp60_(p216) conjugated to PEG.

Expansion of CD8+ Treg cells specific for Qa-1(HLA-E)-Qdm or Qa-1(HLA-E)-Hsp60_(p216) in vivo can also be induced by administering dendritic cells pulsed with either peptide. It has been discovered that immunization with Hsp60_(p216)-loaded dendritic cells (DC) efficiently inhibits the development of collagen-induced arthritis (CIA). Accordingly, aspects of the invention include a method for treating an autoimmune disease. The method comprises contacting dendritic cells isolated from a subject in need of such treatment with Hsp60_(p216) peptide to generate Hsp60_(p216)-loaded dendritic cells; and administering to the subject the Hsp60p216-loaded dendritic cells in an amount effective to ameliorate a symptom of the autoimmune disease.

The term “contacting dendritic cells isolated from a subject in need of such treatment with Hsp60_(p216) peptide” includes any means of contacting including but not limited to, mixing the Hsp60_(p216) peptide and the cells in medium, mixing the Hsp60_(p216) peptide in liposomes with the cells or expressing the Hsp60_(p216) peptide from a recombinant nucleic acid in the cells. In some embodiments, the dendritic cells are contacted with Hsp60_(p216) for 2, 4, 6, 8, 12, 16, 18 or 24 hours to generate Hsp60_(p216)-loaded dendritic cells.

The term “Hsp60_(p216)-loaded dendritic cell” as used herein refers to a dendritic cell presenting Hsp60_(p216) on its surface in a manner effective to selectively expand CD8+ Treg cells that specifically recognize Hsp60_(p216) The dendritic cell may become loaded with the peptide by directly binding the peptide from the medium on its surface or by processing the peptide intra-cytoplasmically before presenting the peptide. Processing the peptide may include proteolytically generating the presented peptide from a longer peptide.

Some aspects of the invention involve a method for treating an autoimmune disease comprising isolating from a subject in need of such treatment CD8+ T cells that bind Hsp60_(p216) peptide; growing the isolated cells in a culture medium containing complexes of IL-15 and IL-15 receptor (IL-15C) until the number of CD8+ T cells that bind Hsp60p216 peptide increases to at least 3-5% of CD8+ T cells, thereby producing a population of cells enriched with CD8+ T cells that bind Hsp60p216 peptide; and administering CD8+ T cells that bind Hsp60p₂₁₆ peptide from the population of cells to the subject in an amount effective to ameliorate a symptom of the autoimmune disease.

Expansion of CD8+ Treg cells specific for Qa-1(HLA-E)-Qdm or Qa-1(HLA-E)-Hsp60_(p216) in vivo can be induced using a moiety comprising at least 4 units of Qa-1(HLA-E)-peptide complex. Accordingly, in some embodiments, the isolating step comprises sorting a sample containing T cells obtained from the subject into CD8+ T cells that bind Hsp60_(p216) peptide using a fluorescently labeled moiety having at least 4 units of a complex, wherein the complex is a major histocompatibility complex (MHC) class I antigen E (HLA-E) linked to Hsp60_(p216) peptide. In some embodiments, the HLA-E-peptide complex comprises biotin, and at least 4 such complexes associate through biotin-avidin interaction to form the moiety. In some embodiments, the avidin is fluorescently labeled. In some embodiments, the avidin is labeled with Phycoerythrin (PE). In some embodiments, the population of cells enriched with CD8+ T cells that bind Hsp60p216 peptide is further enriched using anti-R-Phycoerythrin (PE) microbeads. Specifically, CD8 T cells are first labeled with Qa-1-Hsp60p216 tetramers conjugated with Phycoerythrin (PE). These tetramer positive CD8 cells can then be selected by subsequent labeling with anti-PE antibody conjugated with microbeads and then by mounting them onto the magnetic field. Cells are then collected by detaching from the magnetic field. This procedure enables the enrichment of Tet+ CD8 T cells from the CD8+ T cell pool.

A subject in need of treatment of autoimmune disease is a subject identified as having an autoimmune disease, i.e. the subject has been diagnosed by a physician (e.g., using methods well known in the art) as having an autoimmune disease. In some embodiments, the subject in need of treatment is a subject suspected of having or developing an autoimmune disease, such as a subject presenting one or more symptoms indicative of an autoimmune disease. In some embodiments, a subject suspected of having an autoimmune disease may display abnormal titres of autoantibodies. The subject having abnormal titres of autoantibodies may have at least one other symptom of autoimmune disease or may be without other symptoms associated with autoimmune disease. The term “subject in need of treatment” further includes people who once had an autoimmune disease but whose symptoms have ameliorated.

The subject is an animal, typically a mammal. In one aspect, the subject is a dog, a cat, a horse, a sheep, a goat, a cow or a rodent. In important embodiments, the subject is a human.

A self-antigen (or auto-antigen) is a subject's self-produced constituent, against which the subject mounts an undesired immune response. An “autoantibody” is an antibody produced by a subject, which binds to one or more of the subject's own constituents or self-antigens. The term ‘autoimmune disease’ refers to those disease states and conditions wherein the immune response of the patient is directed against the patient's own constituents resulting in an undesirable and often terribly debilitating condition. As used herein, ‘autoimmune disease’ is intended to further include autoimmune conditions, syndromes and the like. Example of autoimmune diseases include, but are not limited to systemic lupus erythematosus, chronic graft versus host disease, rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis, inflammatory bowel disease, Sjogren's syndrome, Graves disease, Crohn's disease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome, monoclonal gammopathy of undetermined origin, POEMS syndrome, myeloma, macroglobulinemia, and cold agglutinin disease. In some embodiments, the autoimmune disease involves antibodies to a self-antigen and the subject has the antibodies to the self-antigen.

The nanoparticles, Hsp60p216-loaded dendritic cells and the enriched CD8+ T cells that bind Hsp60p216 peptide described herein can also be used to treat human immunodeficiency virus (HIV) infection. CD4+ follicular helper T cells (TFH) are known to serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production (J Exp Med. 2013 Jan. 14; 210(1):143-56). As described herein, the specialized regulatory CD8+ T cells selectively suppress CD4+ follicular helper T cell (TFH) activity through perforin-dependent lysis and elimination by recognition of class Ib major histocompatibility complex (MHC) peptide Qa-1 (mouse homolog of human leukocyte antigen E (HLA-E)) expressed at the surface of TFH cells. It is believed that the CD8+ Treg cells specific for Qa-1(HLA-E)-Hsp60_(p216) target activated, HIV-infected TFH cells which present Hsp60p₂₁₆ on their surface. Thus, CD8+ cells specific for Hsp60p₂₁₆ peptide can be used to eliminate the TFH cells that serve as major reservoirs for HIV-1 infection. It is believed that the CD8+ Treg cells specific for Qa-1(HLA-E)-Hsp60_(p216) do not generally suppress the immune response, and instead, target activated, HIV-infected CD4+ follicular helper T cells (TFH) that present the Hsp60_(p216) peptide on their surface. Accordingly, aspects of the invention involve methods of treating HIV infection relate to the development of strategies based on in vivo and in vitro expansion and activation of the CD8+ Treg cells for the treatment of HIV infection.

A subject in need of treatment of HIV infection is a subject identified as having HIV infection, i.e. the subject has been diagnosed by a physician (e.g., using methods well known in the art) as having HIV infection. In some embodiments, the subject in need of treatment is a subject suspected of having or developing HIV infection, such as injection drug users who share needles, infants born to mothers with HIV infection who did not receive HIV therapy during pregnancy, people who received blood transfusions or clotting products between 1977 and 1985 (before screening for the virus became standard practice), people who have unprotected sex, especially with people who have other high-risk behaviors, are HIV-positive, or have AIDS. The term “subject in need of treatment” further includes people who were previously diagnosed with HIV infection and have started to present one or more symptoms of HIV infection.

The subject is an animal, typically a mammal. In one aspect, the subject is a dog, a cat, a horse, a sheep, a goat, a cow or a rodent. In important embodiments, the subject is a human.

The nanoparticles, Hsp60p216-loaded dendritic cells and the enriched CD8+ T cells that bind Hsp60p216 peptide described herein are administered in an effective amount to treat autoimmune diseases or HIV infection. An effective amount is a dose sufficient to provide a medically desirable result and can be determined by one of skill in the art using routine methods. In some embodiments, an effective amount is an amount which results in any improvement in the condition being treated. In some embodiments, an effective amount may depend on the type and extent of the autoimmune disease or condition being treated and/or use of one or more additional therapeutic agents. However, one of skill in the art can determine appropriate doses and ranges of therapeutic agents to use, for example based on in vitro and/or in vivo testing and/or other knowledge of compound dosages.

When administered to a subject, effective amounts of the therapeutic agent will depend, of course, on the particular disease being treated; the severity of the disease; individual patient parameters including age, physical condition, size and weight, concurrent treatment, frequency of treatment, and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In some embodiments, a maximum dose is used, that is, the highest safe dose according to sound medical judgment.

In the treatment of autoimmune disease, an effective amount is that amount which slows the progression of the disease, halts the progression of the disease, or reverses the progression of the disease. An effective amount includes that amount necessary to slow, reduce, inhibit, ameliorate or reverse one or more symptoms associated with the autoimmune disease. In some embodiments, such terms refer to a reduction in the swelling of one or more joints or a reduction in the pain, fatigue and/or fever associated with an autoimmune disorder. In some embodiments, such terms refer to a reduction in the levels of circulating autoantibodies associated with the autoimmune disease. In some embodiments, such terms refer to a reduction in a human's PASI score. In some embodiments, such terms refer to an improvement in a human's global assessment score.

In the treatment of HIV infection, an effective amount is that amount which slows the progression of the disease, halts the progression of the disease, or reverses the progression of the infection. An effective amount includes that amount necessary to slow, reduce, inhibit, ameliorate or reverse one or more symptoms associated with the HIV infection.

An effective amount of a compound typically will vary from about 0.001 mg/kg to about 1000 mg/kg in one or more dose administrations, for one or several days (depending of course of the mode of administration and the factors discussed above).

Actual dosage levels of the therapeutic agent can be varied to obtain an amount that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level depends upon the activity of the particular compound, the route of administration, the tissue being treated, and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effort and to gradually increase the dosage until the desired effect is achieved.

Pharmaceutical preparations and compounds are administered to a subject by any suitable route. For example, compositions can be administered orally, including sublingually, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically and transdermally (as by powders, ointments, or drops), bucally, or nasally. The pharmaceutical preparations of the present invention may include or be diluted into a pharmaceutically-acceptable carrier. The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible fillers, diluants or other such substances, which are suitable for administration to a human or other mammal such as a dog, cat, or horse. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The carriers are capable of being commingled with the preparations of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy or stability. Carriers suitable for oral, subcutaneous, intravenous, intramuscular, etc. formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

The present invention is further illustrated by the following Example, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

Example

A small subset of CD8 cells is essential for the maintenance of self-tolerance and plays an important role in the inhibition of autoimmune disease 1. This subset of regulatory T cells recognizes Qa-1-peptide complexes that are upregulated by pathogenic CD4 T cells, resulting in elimination of these cells through perforin-dependent lysis. Development of strategies based on in vivo expansion and activation of CD8+ Treg represents a new avenue of immunotherapy for the treatment of autoimmune diseases.

Antigen Presentation by Qa-1 (HLA-E):

Two major peptides presented by Qa-1 (HLA-E) are Qdm (B7sp in human) and Hsp60p216. Qdm (B7sp) is a peptide derived from the leader sequence of MHC class Ia which can bind to both the TCR and to CD94/NKG2A receptors. The Hsp60_(p216) peptide can efficiently replace Qdm peptides under conditions of cellular activation and stress. A third peptide that is presented by Qa-1 under conditions of defective antigen processing in the endoplasmic reticulum (ERAAP dysfunction) is the highly conserved peptide FL9. Recognition of Qa-1/FL9 by CD8 T cells may serve to eliminate abnormal cells that have defects in antigen presentation. In sum, Qa-1 dependent recognition represents a potential screening system for cells that are stressed or activated, as demonstrated in cases of infection and secondary to cellular defects of peptide processing associated with autoimmunity and cellular transformation.

Qa-1-peptide tetramers have been generated to detect peptides-specific CD8+ T cells. In the case of Qa-1-Qdm tetramers, a Qa-1 protein containing a point mutation (R72A) was used to prevent binding of Qa-1-Qdm to CD94/NKG2A receptors and allow specific detection of CD8+ T cells that express Qa-1 restricted TCR.

DC/Peptide Immunization and Inhibition of Autoimmune Disease:

Expansion of CD8 T cells specific for Qa-1-Qdm or Qa-1-Hsp60_(p216) in vivo can be induced by vaccination of mice with dendritic cells pulsed with either peptide. WT B6 mice that were immunized with chicken collagen type II were treated by subcutaneous injection of irradiated B6. Kb^(−/−)Db^(−/−)DC pulsed with either Hsp60_(p216) or Qdm peptides at days 27, 36, and 49 and the progression of arthritis was monitored. Immunization with Hsp60_(p216)-loaded DC efficiently inhibits the development of collagen-induced arthritis (CIA), while vaccination with DC pulsed with Qdm peptide does not suppress disease progression (FIG. 1A). Immunization of mice with Hsp60_(p216)-loaded Kb^(−/−)Db^(−/−)DC was associated with expansion of Qa-1-Hsp60_(p216)-specific CD8+ T reg cells as detected by Qa-1-Hsp60_(p216) tetramers (FIG. 1B). Although immunization with Qdm-loaded Kb^(−/−)Db^(−/−)DC resulted in increased numbers of Qa-1 R72A-Qdm tetramer+ cells (FIG. 1B), this immunization did not inhibit disease (FIG. 1A).

These findings indicate that specific in vivo expansion of Hsp60p216-specific CD8 Treg can contribute to the suppression of pathogenic CD4 cells and inhibition of disease progression, a finding supported by adoptive transfer studies of purified tetramer+ Hsp60_(p216)-specific CD8+ T cells as described below.

Inhibition of Autoimmune Disease by Qa-1-Hsp60p216 Tetramer+ CD8 Cells:

To test the ability of Hsp60_(p216)-Qa-1-restricted CD8 cells to carry CD8+ T reg activity, Qa-1-Hsp60_(p216)-tetramer+ CD8 T cells were separated from the tetramer-fraction by consecutive FACS sorting and microbead selection (FIG. 2A). Adoptive transfer of enriched Qa-1-Hsp60_(p216)-tetramer+ CD8 cells (3.5×10⁴ cells), but not Qa-1-Hsp60_(p216)-tetramer− CD8 cells into Rag2−/−Prf1−/− hosts along with CII-immune CD4 and B cells inhibited autoantibody production and halted arthritis progression (FIG. 2B-D). Analysis of CD8 cells in these adoptive hosts revealed an expanded population of Qa-1-Hsp60_(p216)-tetramer+ CD8 cells after transfer of tetramer+ but not after transfer of tetramer− CD8 cells (FIG. 2E). In contrast, transfer of R72A Qa-1-Qdm tetramer+ CD8+ cells did not prevent disease progression, consistent with the failure of Qdm-DC immunization to inhibit disease progression.

Effects of p-HLA-E-NP Vaccination in Humanized Mice:

Expansion of HLA-E/peptide specific CD8 cells by p-HLA-E-NP vaccination is tested in NOD.Cg-B2m^(tm1Unc) Prkdc^(scid) Il2rg^(tmlWjl)/SzJ (NSB) mice expressing the HLA-E transgene (NSB-HLA-E) covalently linked to h132m using HLA-E/peptide tetramers. Humanized NSB-HLA-E mice that are reconstituted with human immune cells and vaccinated with p-HLA-E-NP are tested for the frequency of peptide specific human CD8 cells and their surface phenotype (CD45RO and KIR). Monitoring of the expansion of HLA-E/HSP_(p216) specific CD8 T cells by detecting these cells using tetramers in humanized mice can be extended to detection of Tetramer⁺ CD8 cells in PBMC of patients.

Superior Feature of p-Qa-1 (HLA-E)-NP Vaccination:

Both Qa-1 in mice and HLA-E in men exhibit a limited polymorphism distinct from MHC class Ia molecules that are highly polymorphic. The human HLA-E gene, for example, is expressed one out of two alleles which differ at a single amino acid. Therefore, a generalized immunotherapy using p-Qa-1 (HLA-E)-NP-based vaccination for the treatment of autoimmune disease represents a promising therapeutic strategy that does not depend on extensive individualized pMHC-NP design.

Qa-1 R72A-Qdm and Qa-1-Hsp60_(p216) tetramers were designed to detect CD8⁺ T cells specific for Qa-1-Qdm or Qa-1-Hsp60_(p216). The tetramers were used to enrich CD8 cells specific for these ligands and the cells were tested for regulatory activity in the context of CIA. Vaccination of mice with dendritic cells (DC) pulsed with defined peptides, resulted in expansion of CD8⁺ T cells specific for Qa-1-Qdm or Qa-1-Hsp60_(p216) in vivo. Although immunization with Hsp60_(p216)-loaded DC efficiently inhibited the development of CIA, vaccination with DC pulsed with Qdm peptide did not suppress disease progression. Moreover, transfer of a small number of Qa-1-Hsp60p216 tetramer⁺ CD8⁺ cells, but not R72A Qa-1-Qdm tetramer⁺ CD8⁺ cells, inhibited disease progression. Altered processing of MHC class Ia leader peptide in activated cells or stressed cells results in increased processing and presentation of alternate peptides, including those derived from Hsp60, to Qa-1-restricted CD8⁺ T cells. Inhibitory signaling associated with the interaction between the CD94/NKG2A receptor and the Qa-1-Qdm complex overrides TCR-dependent activation by Qa-1-Qdm ligands.

In sum, amelioration of autoimmune arthritis may be achieved by targeting arthrogenic T_(FH) and T_(H)17 cells through mobilization of CD8⁺ Treg. CD8⁺ Treg infusion may be used for patients who develop resistance to MTX treatment or relapse after MTX withdrawal. Moreover, the IL-15 dependence of CD8⁺ Treg may be exploited for development of personalized CD8⁺ Treg-based cellular therapy to arthritis after in vitro expansion, or direct peptide-based expansion of CD8⁺ Treg in vivo. The development of strategies based on specific expansion and activation of CD8⁺ Treg represents a new and potentially effective approach to the treatment of autoimmune disease.

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the above description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

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We claim:
 1. A nanoparticle comprising a biocompatible polymer and a complex, wherein the complex is a major histocompatibility complex (MHC) class I antigen E (HLA-E) linked to a peptide, and wherein the HLA-E-peptide complex is linked to the surface of the nanoparticle.
 2. The nanoparticle of claim 1, wherein at least 4 units, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 units of the HLA-E-peptide complex are linked together to form a moiety and the moiety is linked to the surface of the nanoparticle.
 3. (canceled)
 4. The nanoparticle of claim 1, wherein the peptide is selected from the group consisting of Hsp60_(p216), B7sp and FL9.
 5. (canceled)
 6. The nanoparticle of claim 1, wherein the peptide is linked to the HLA-E via a flexible linker.
 7. (canceled)
 8. The nanoparticle of claim 1, wherein heavy chain and light chain (β-2 microglobulin) of the HLA-E are linked via a flexible linker. 9-10. (canceled)
 11. The nanoparticle of claim 1, wherein a low releasing dose of IL-15 is incorporated into the nanoparticle.
 12. A method for treating an autoimmune disease comprising: administering to a subject in need of such treatment nanoparticles of claim 1 in an amount effective to ameliorate a symptom of the autoimmune disease.
 13. (canceled)
 14. A method for treating an autoimmune disease comprising: contacting dendritic cells isolated from a subject in need of such treatment with Hsp60_(p216) peptide to generate Hsp60_(p216)-loaded dendritic cells; and administering to the subject the Hsp60_(p216)-loaded dendritic cells in an amount effective to ameliorate a symptom of the autoimmune disease. 15-16. (canceled)
 17. A method for treating an autoimmune disease comprising: (a) isolating from a subject in need of such treatment CD8+ T cells that bind Hsp60_(p216) peptide; (b) growing the isolated cells in a culture medium containing IL-15C until the number of CD8+ T cells that bind Hsp60_(p216) peptide increases to at least 3-5% of CD8+ T cells, thereby producing a population of cells enriched with CD8+ T cells that bind Hsp60_(p216) peptide; and (c) administering CD8+ T cells that bind Hsp60_(p216) peptide from the population of cells to the subject in an amount effective to ameliorate a symptom of the autoimmune disease.
 18. The method of claim 17, wherein isolating in step (a) comprises sorting a sample containing T cells obtained from the subject into CD8+ T cells that bind Hsp60_(p216) peptide using a fluorescently labeled moiety having at least 4 units of a complex, wherein the complex is a major histocompatibility complex (MHC) class I antigen E (HLA-E) linked to Hsp60_(p216) peptide.
 19. The method of claim 18, wherein the HLA-E-peptide complex comprises biotin, and at least 4 such complexes associate through biotin-avidin interaction to form the moiety. 20-23. (canceled)
 24. A method for treating HIV infection comprising: administering to a subject in need of such treatment nanoparticles of claim 1 in an amount effective to ameliorate a symptom of the HIV infection.
 25. A method for treating HIV infection comprising: contacting dendritic cells isolated from a subject in need of such treatment with Hsp60_(p216) peptide to generate Hsp60_(p216)-loaded dendritic cells; and administering to the subject the Hsp60_(p216)-loaded dendritic cells in an amount effective to ameliorate a symptom of the HIV infection.
 26. (canceled)
 27. A method for treating HIV infection comprising: (a) isolating from a subject in need of such treatment CD8+ T cells that bind Hsp60_(p216) peptide; (b) growing the isolated cells in a culture medium containing IL-15C until the number of CD8+ T cells that bind Hsp60_(p216) peptide increases to at least 3-5%, of CD8+ T cells thereby producing a population of cells enriched with CD8+ T cells that bind Hsp60_(p216) peptide; and (c) administering CD8+ T cells that bind Hsp60_(p216) peptide from the population of cells to the subject in an amount effective to ameliorate a symptom of the HIV infection.
 28. The method of claim 27, wherein isolating in step (a) comprises sorting a sample containing T cells obtained from the subject into CD8+ T cells that bind Hsp60_(p216) peptide using a fluorescently labeled moiety having at least 4 units of a complex, wherein the complex is a major histocompatibility complex (MHC) class I antigen E (HLA-E) linked to Hsp60_(p216) peptide.
 29. The method of claim 28, wherein the HLA-E-peptide complex comprises biotin, and at least 4 such complexes associate through biotin-avidin interaction to form the moiety. 30-32. (canceled) 